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Following is a list of categories providing possible examples of procedures which are representative of each category.

Type A: Studies which cause little or no pain or distress.
Examples include housing and brief restraint of animals for observation or examination; single blood sampling; single injections of non–toxic materials; standard approved methods of euthanasia that induce rapid unconsciousness; short periods (a few hours) of food and water deprivation; and behavioral observations.

Type B: Studies which may involve minor pain or distress of short duration.
These include surgical procedures and other studies on anesthetized animals where the animals do not regain consciousness (eg non–survival surgery). Also included are cannulation of vessels or body cavities performed under anesthesia; minor surgical procedures such as biopsies and others where post–surgical pain and distress are minimal; overnight or longer food or water deprivation; behavioral studies on awake animals that involve short–term restraint; studies using noxious stimuli from which escape is possible; using tumor implants or hybridomas under guidelines as outlined by the IACUC; and the use of Freund’s complete adjuvant injected intramuscularly.

Comment: During and after Type B studies animals are not expected to show anorexia, dehydration, abnormal discharges, hyperactivity, increased recumbency or dormancy, increased vocalization, self–mutilation, aggressive–defensive behavior or demonstrate social withdrawal and self–isolation.

Type C: Studies which may involve moderate to severe pain or distress.
These include major surgical procedures performed under anesthesia, permitting recovery, with adherence to acceptable veterinary practices including postoperative analgesia, fluid therapy and veterinary nursing care; prolonged periods (several hours or more) of physical restraint or deprivation of the animals’ environmental necessities, such as maternal deprivation, aggression, predatory–prey interactions; procedures which alter perceptual or motor functions, such as the induction of paralysis or seizures; and induction of infectious diseases or toxicities, and when severe clinical symptoms begin to appear the animals are treated or euthanized.

Comment: Involvement of trained technicians, scientists and veterinarians is critical if this pain is to be minimized or avoided. Animals used in Type C studies should not show signs of prolonged clinical distress, such as behavioral abnormalities, lack of grooming, dehydration, anemia, abnormal vocalization, prolonged anorexia, self-mutilation, increased signs of infectious processes (peritonitis, pneumonia, diarrhea, encephalitis, etc.). If these clinical abnormalities develop, the necessary treatments to alleviate the symptoms must be available and provided. If the symptoms cannot be alleviated, the animals must be euthanized with minimal delay.

Type D: Projects that may involve moderate to severe pain or distress without the benefit of pain–relieving drugs or other appropriate therapy.
Such studies include application of noxious stimuli from which escape is impossible; exposure to noxious stimuli or agents whose effects are unknown; completely new experiments which have a high degree of invasiveness; induction of aggressive behavior leading to self–mutilation or fighting; and induction of infectious diseases or toxicities where death is an end point and animals are not treated or euthanized when severe clinical abnormalities develop.

Comment: Type D projects present an explicit responsibility on the faculty to explore alternative methods before proceeding with the study. Type D projects are considered by some to be highly questionable or unacceptable, irrespective of the significance of the anticipated results. Before our Committee can review and approve these projects, the justification statements and the veterinary involvement must be clearly presentable and understandable.

All personnel (faculty, staff, students) who will use animals on the protocol must be listed with either their social security number or USC employee number. The USC security department requires one of these numbers for identification of staff who will work in animal facilities after hours.

The training status of all staff must also be listed. Two types of training are required:

  • Animal Resources training in proper animal care, handling and experimental manipulation.
  • Occupational health and safety training offered by the Department of Safety and Risk Management.

Both types of training must be completed by all personnel. Completion of training will be listed as a required stipulation of IACUC approval if it has not already been completed for the personnel listed.

Example 1:

The purpose of the present proposal is to examine the role of metabolic factors, specifically those independent of insulin (termed “glucose effectiveness”), in the development of glucose intolerance of normal aging. These studies will aid our understanding of the cellular and tissue–specific processes involved in glucose effectiveness, and the impact of the dietary changes in the amelioration of age associated glucose intolerance.

One of the metabolic characteristics of advanced age in humans and animals is the impaired ability of the body to dispose of carbohydrates. This impairment, termed “glucose intolerant”, results in increased mortality even in the absence of overt diabetes. Along with steady increments of fasting blood sugar with age, prolonged elevation of the blood sugar may lead to biochemical “attachment” of sugar to various body proteins, a process implicated in age–associated complications such as atherosclerosis and glaucoma. While insulin is the major hormone which regulates glucose tolerance, factors independent of insulin, (“termed glucose effectiveness”), are equally important, and effectiveness is extremely important, since reduced glucose effectiveness is a known risk factor for development of diabetes.

The primary objective of this proposal is to investigate the role of glucose effectiveness in the development of the glucose intolerance of aging. By understanding the mechanism by which the glucose intolerance develops with advanced age, it may be possible to avert the development of age–associated metabolic complications.

Example 2:

We are investigating a non–invasive technique for the detection of skin cancer/melanoma. A short duration, low energy laser pulse is applied to both cancerous and normal tissue and measurements are taken to quantify how much of the energy is absorbed by the tissues. By comparing measurements taken lesions and those taken from normal skin, we hope to identify a pattern that will be indicative of disease. Our ultimate goal is to develop a system for clinical application as a non-invasive method of detecting melanoma, without having to perform a biopsy of the lesion.

Example 3:

The primary goal of the proposed work is to increase understanding of the initial molecular sites and mechanism(s) causing ethanol’s behavioral effects. The proposal is investigating ethanol’s action on a family of receptors called ligand gated ion channels (LGICs) that mediate communication between nerve cells. The proposal builds on previous findings demonstrating that increased atmospheric pressure (Hyperbaric exposure) is a direct, highly sensitive antagonist of ethanol’s behavioral and biochemical effects. The proposed studies will use the unique physico–chemical properties and selectivity of pressure antagonism, in combination with behavioral, recombinant and electrophysiological approaches, to address key questions regarding the way alcohol works in the brain. The use of increased atmospheric pressure in this way represents an innovative use of a physical manipulation to achieve end results that cannot be achieved using traditional pharmacological antagonists or other currently available techniques. The pattern of the mechanism(s) and sites of action on LGICSs and, in turn, will aid in the development of molecular models of ethanol’s sites of action. Overall, the proposed work will contribute to the long term goal, which is to identify specific targets at which therapeutically relevant agents can be directed to reduce the social problems, loss of lives and tremendous economic costs resulting from the misuse and abuse of alcohol.


Because of the variable nature of animal research at this University we cannot offer specific rules for justification. However, the experimental group size and numbers of experimental groups must be explained and a logical mathematical progression to produce the final total of animals required must be supplied. General guidelines and examples are as follows:

If applicable, provide justification for the total number of animals used or produced, not just the number of animals from which data will be collected. In the case of a breeding colony, list the number of breeding animals to be obtained, the total number of offspring born, and the proportion of these actually used for experiments (if all genotypes produced are not usable). For example: “We will introduce 12 breeding animals (6 pairs) of the genetic line into our colony room and produce an estimated 120 mouse pups from the colony. Of these, only 25% will be homozygous recessive to supply the estimated 40 mice for tissue collection as described.”

If applicable, include in your justification a consideration of the number of animals that can be expected to be lost to failure of a procedure. For example: “Although we are requesting 8 animals per group, in our experience 20% of the animals will be lost due to improper probe placement. Therefore, we have increased the total number of animals requested from 400 to 480.”

Provide justification for the actual number of animals to be used for the experimental procedures. Valid types of justifications include:

Statistical significance. Name the statistical analysis that will be used and the level of significance used to test the hypothesis. You must provide evidence that the number of animals you are requesting will produce a significant test. This evidence can come from personal experience with the species and system, the experience of other investigators, or a power analysis. For example: “Our previous experience indicates that 8 animals per group will provide sufficient statistical strength in these studies to detect significant differences at the p>0.05 level with ANOVA. Since we have 5 groups in each experiment and propose to do ten experiments as detailed in the narrative, we are requesting 400 animals (8x5x10).”

A specific quantity of tissue, antibodies or cells is needed to complete the study. Explain why this quantity is needed and justify the number of animals that will be used to reach this quantity. For example: “For each year of the studies detailed in this proposal, we will require 600 mg. of striatal tissue. Each rat striatum weighs approximately 50 mg. Therefore, 12 animals per year are necessary to obtain the needed tissue.”

Pilot study. If you have limited experience with a system such that trial experiments are needed for training or assessment of feasibility, or for any reason you are unable to accurately estimate the number of animals that will be needed to complete the study, you may request a relatively small number of animals for a pilot study. At completion of the pilot study you will be required to submit a separate and final protocol to the IACUC for review in which the total number of animals is adequately justified. For example: “Before performing more detailed studies, we need to determine whether the transgenic mice are producing the protein of interest. To determine this, we must analyze the livers from five transgenic and five control mice. We are requesting only ten mice at this time and will submit a separate protocol form for future studies if the protein of interest is expressed.”

If transgenic animals will be used on the protocol, this section of the form must be filled out. If the transgenic animals will be produced by another laboratory or institution, the animal procedures used to produce the animals (superovulation, embryo collection, embryo transfer) should not be listed on the protocol form. The source which will produce and supply the animals should be listed. However, the information regarding potential adverse effects and monitoring for adverse outcomes must be included for all protocols in which transgenic animals are to be used.

Federal law (Animal Welfare Act, 7 U.S.C. 2121, et seq.) requires written investigator assurance “. . . that alternatives to procedures that may cause more than momentary or slight pain or distress have been considered and written description of the methods and sources used to determine that alternatives are not available.” 

Therefore, protocols must include this assurance and written description of the methods and sources consulted prior to approval by the IACUC. A search for alternatives to potentially painful procedures must be conducted. Searches should be conducted using databases such as Medline, AWIC, CRIS, Index Medicus, etc. The minimal written narrative should include: the databases searched or other sources consulted, the date of the search and the years covered by the search. At least two databases must be searched, although databases containing multiple sub–databases (e.g. Ovid search) are also acceptable. A protocol will not be approved without this information.

The IACUC requires the original signature of the principal investigator for the protocol to be approved. The principal investigator must be a USC faculty member. Signatures of coinvestigators and other staff will not be accepted. The signature indicates the person with overall responsibility for the protocol and animals used on the protocol. If a coinvestigator will assume this responsibility, the entire protocol should be in their name with their signature. Please follow the link to the E-Signatures in iStar Reference Guide.

To kindly assist all our principal investigators and their approved personnel, the iStar IACUC PI Reference Guide will provide step by step instructions in submitting an IACUC protocol. In addition how to assign Authorized Submitters, addressing stipulations, submitting Amendments and Renewals, and withdrawing submissions.